Cilia are organelles found on most cells where they play vital signaling and motility roles required for normal human development and normal organ function. Defects in ciliary assembly or function can lead to multiple human pathologies, called ciliopathies, including developmental defects, polycystic kidney disease, several syndromes and primary ciliary dyskinesia (PCD). The focus of the proposed studies is on dynein-driven ciliary movement. Failure in assembly or regulation of the dyneins motors can result in PCD, however, we are only beginning to understand how the ciliary dynein motors are regulated. The major questions addressed in this study include: What is the mechanism that ensures ciliary bends are initiated and propagated from the base? How are ciliary beat frequency and waveform regulated? How is the activity of the multiple dynein motors coordinated? How do changes in axonemal phosphorylation regulate ciliary bending? Aim 1 addresses the question of how bending is initiated at the base of the axoneme. This aim is based on new results indicating that the proximal 2 um of the axoneme is exceptionally stable, a feature that may be critical for initiating ciliary bending from the base of the axoneme. Experiments are designed to identify novel proteins specifically assembled at the base of the axoneme and to test the hypothesis that specialized dyneins localized to the proximal axoneme perform in an unconventional manner to maintain strong physical linkage between the outer doublet microtubules. In Aims 2 and 3, experiments are designed to determine the mechanisms that regulate dynein by phosphorylation and control the size and shape of the ciliary bend, parameters that define ciliary waveform. The studies are founded on discovery that the conserved ciliary dynein, I1 dynein, is regulated by changes in phosphorylation of the intermediate chain IC138 and important for control of ciliary waveform. Aim 2, uses novel cryo electron tomography and molecular approaches to define the structural and functional interaction between I1 dynein and the newly identified and conserved dynein regulator called the Mia complex. In parallel, this study will also directly test the role of the regulatory phosphoprotein for regulation of I1 dynein and ciliar bending. Aim 3 also takes advantage of molecular approaches and cryo electron- tomography to localize key signaling components, CK1 and PP2A, required to control dynein activity and ciliary waveform. This study takes advantage of the highly ordered structure of the axoneme to define general principles for localizing otherwise ubiquitous kinases (CK1) and phosphatases (PP2A), in this case predicted to localize near the base of I1 dynein. As a complementary approach, novel suppressor mutants of pf4, a mutant defective in PP2A assembly, will be characterized to define how PP2A regulates ciliary bending. These studies address fundamental questions about initiation and control of ciliary bending and how conserved, ubiquitous kinases and phosphatases are localized to regulate ciliary bending. Predictably new genes identified will encode proteins that, when defective, will result in PCD.